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The effect of short term incubation in vitro on the RNA content of regenerating mouse epidermis.

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THE ANATOMICAL RECORD 196~349-353 (1980)
The Effect of Short Term Incubation In Vitro
on the RNA Content of Regenerating Mouse
Epidermis
MARGARET ANNE TORMAY AND THOMAS S. ARGYFtIS
Department of Pathology, Upstate Medical Center, SUNY, Syracuse, New York
ABSTRACT
The effect of short term incubation in vitro on RNA content in
regenerating and normal epidermis has been investigated. Regenerating mouse
epidermis was incubated for three hours, either attached to its underlying dermis
or by itself, i n either buffered sucrose or Roswell Park Memorial Institute culture
medium at 26°C or at 37°C. There is a significant loss of RNA when regenerating
epidermis is incubated without being attached to its underlying dermis, a t either
26°C or 37"C, although there was little loss of DNA, good incorporation of ("HI
orotic acid into RNA, as well as good preservation of epidermal histological details.
In contrast, when regenerating epidermis was incubated attached to its dermis,
little loss of RNA occurred. Similarly, incubating normal epidermis attached to its
dermis results i n no loss of RNA. These conditions also result in no significant loss
of DNA, good incorporation of ("HI orotic acid into RNA, and preservation of
epidermal histological details.
The study of epidermal RNA synthesis i n
vitro has increased considerably (Flamm et al.,
'66; Delapp and Karasek, '76). Usually much
attention is paid to make certain that there is a
linear incorporation of the appropriate precursors into RNA (Flamm et al., '66; Delapp
and Karasek, '76) under the conditions of study
in vitro. To the best of our knowledge, no attention has been paid to whether or not the in vitro
incubation of the epidermis results in a loss of
RNA and if so, if i t is related to a change in the
histological appearance of the epidermis.
Because we have been interested in the role
of the RNA, which accumulates during epidermal regeneration in the regulation of the epidermal hyperplastic growth (Argyris, '76; '77;
'78), and in skin carcinogenesis (DeYoung et
al., '77; Mueller and Argyris, '77), we have been
seeking conditions for the short term study of
RNA metabolism of the hyperplastic epidermis
in vitro. This report will demonstrate that the
incubation of regenerating epidermis for as little as three hours in vitro can result in the loss
of considerable RNA, even though there is a
vigorous incorporation of precursor into RNA.
Moreover, the loss of the RNA is not reflected in
any change in the histological appearance of
the regenerating epidermis. We will also show
that under some incubation conditions, we can
prevent the loss of much of the RNA if the
000-3276X/80/1963-0349$01.400 1980 ALAN R. LISS, INC.
regenerating mouse epidermis is left attached
to its dermis.
MATERIALS AND METHODS
Mice and abrasion technique
CD-1 female mice, 40 days of age, were purchased from Charles River Farms, Wilmington,
Mass. The conditions for maintenance of the
mice has been already described (Argyris, '76;
'77). Epidermal abrasion was performed by
using a felt wheel mounted on a Dremel mototool as previously described (Argyris, '76; '77).
Media
Sucrose. 0.25 M was made in 0.02 M Trizma
(pH 7.5, 20°C), 0.25 M KC1, 0.005 M MgC12,
and 0.001 M dithiothreitol (STKM). Roswell
Park Memorial Institute tissue culture medium 1640 (RPMI) was obtained from Dr. Bertie F. Argyris, Department of Microbiology in
this institution. The contents of the medium
are described in Argyris, B. ('77), except we did
not add serum to the medium.
Preparation of epidermis for in uitro study
Regenerating epidermis was studied in vitro
in two different ways. It was placed in vitro,
attached to its underlying dermis (epidermis
Received June 21, 1979, accepted September 4, 1979
350
MARGARET ANNE TORMAY AND THOMAS S. ARGYRIS
plus dermis), or separated from its underlying
dermis (epidermis alone).
For the incubation in vitro of regenerating
epidermis with its dermis, 8 abraded mice were
sacrificed by cervical dislocation. The backs
clipped, the depilatory Surgex applied for two
minutes and rinsed off with running water (Argyris, '77). The abraded area was cut out and
placed in saline at 4°C. The subcutaneous tissues were scraped away (Argyris, '77) and the
regenerating epidermis, along with its underlying dermis, was placed i n either 0.25M
STKM or RPMI, in plastic tissue culture dishes,
60 x 15 mm (Falcon Plastics, Los Angeles,
CA.). Usually, two wounds were divided in half,
each piece was about 1.5 cm2, and the four
pieces were placed in a plastic culture dish.
Each piece contained about 12 mg of epidermis.
For each determination, the epidermis from the
4 halves was pooled. The incubation period was
for 3 hours, either at room temperature (26"C),
or at 37"C, in a CO, incubator. To the medium,
25 pCi/ml of (5-"H)orotic acid (Schwarz/Mann,
Orangeburg, N.Y.) Sp.Act. 10 Ci/mM, at a concentration of 0.5 mCi/ml, was added. Total volume of the incubation mixture was 4 ml. Following the 3 hours incubation period, the
wounds were removed and washed 3 times with
ice cold saline. The regenerating epidermis was
then scraped off with a scalpel, dried between 2
layers of No. l W h a t m a n filter paper, and
weighed (Argyris, '77).
For incubation of regenerating epidermis
alone in vitro, wounds were cut out from mice,
and the subcutaneous tissues removed by
scraping as described above. The wounds were
then placed dermis side down on top of a n ice
filled Petri dish, and the regenerating epidermis scraped off as previously described (Argyris, '77). The epidermis from 2 wounds was
quickly washed in 3 changes of ice cold saline
and placed in a Falcon dish with the appropriate medium. Upon completion of the incubation
period, the regenerating epidermis was removed, rinsed with 3 changes of ice cold saline,
and dried as described above.
Normal epidermis attached to its underlying
dermis was also incubated in vitro for 3 hours a t
26"C, either in 0.25 M STKM or RPMI, with 25
pCi/ml of (5-"H)orotic acid. Total volume of the
incubation mixture was also 4 ml. Four pieces
of normal epidermis plus dermis, cut to approximate the size of the pieces of regenerating epidermis plus dermis, were placed in each dish.
Each piece had about 8 mg of epidermis. The
epidermis from the 4 pieces was pooled for each
determination.
Preparation of homogenates
Epidermal homogenates 4% w/v i n 0.25 M
STKM were prepared before or after incubation
as already described (Mueller and Argyris, '75).
Acid soluble material (ASM), RNA, and DNA
were obtained by extracting 1 m l of t h e
homogenates using a Schmidt-Thannhauser
procedure (Mueller and Argyris, '75). RNA was
measured using lAZBO
units = 32 pgRNA/ml
(Argyris, '78). DNA was determined by measuring the A,,,, using purified calf thymus DNA
(Sigma Chemical Co., St.Louis, MO.) as a standard. The values were similar to those obtained
using the diphenylamine reaction (Argyris,
'78).
The amount of radioactivity in the epidermal
homogenates and in the ASM and RNA fractions from the Schmidt-Thannhauser extraction was determined by placing 0.2 ml of the
homogenate or of the ASM and RNA fractions
i n a scintillation vial, to which was added 1ml
of NCS solubilizer (AmershamiSearle, Arlington Heights, IL.) and 1 0 m l of the premixed
scintillation cocktail, Omnifluor (New England
Nuclear, Boston, Mass.) dissolved in reagent
grade toluene. Sample counting was done in a
Packard Tri-Carb scintillation spectrometer,
model 3320. Counting efficiency was 3Wo.
Background ranged from 11-22 cpm.
Biopsy specimens of epidermis and dermis,
or epidermis alone, from normal and abraded
skin were taken before and after the 3-hour
incubation period, fixed in b d e r e d formalin,
and processed for routine histological study
(Argyris, '76).
Statistical analysis was done using Student t
test. P values of 0.05 or less were considered
significant.
RESULTS
We first have determined whether or not the
3-hour incubation period results in a net loss of
epidermal RNA, DNA, and acid soluble material (ASM). Table I indicates the values for
RNA, DNA, and ASM of regenerating epidermis in intact normal mice. Epidermis incubated alone in vitro at 26°C shows a significant
decrease in RNA. The greatest loss of RNA is in
epidermis incubated alone a t 37°C in RPMI
(Table 2). The least loss of epidermal RNA in
vitro is seen (Table 2) in epidermis plus dermis
incubated at 26°C i n STKM, which shows a n
RNA value which is 94%of that in intact mice.
Incubation of epidermis plus dermis i n RPMI
also results in only a small, but not significant
loss of RNA (Table 2). The concentration of the
ASM increases moderately over that which ap-
35 1
EPIDERMAL RNA IN VITRO
TABLE 1. RNA, DNA, and acid soluble material (ASMI of normal and regenerating
female mouse epidermis
~~
RNA
ASM
(A,,, unitsig epidermis)
(6)"
1.65 i 0.035
2.98 t 0.14
(6)
4.54 2 0.076
34.1 2 4.22
(6)
28.8 & 2.12
(8)
(7)
(9)
(mgi&$ZFmis)
Abraded t
7 days
Normal
~~
DNA
(mgigmis)
4.66
&
0.22'
*Average t standard error of the mean.
"umber of determinations in parentheses.
pears in vivo, depending on the conditions of
incubation (Table 2). The DNA content is not
significantly affected i n any of the incubation
conditions (Table 2). Histological preservation
is quite good under all conditions of incubation
(not shown).
The incorporation of (")
orotic acid into
RNA and ASM is indicated in Table 3. It is clear
that the best incorporation of ("HIorotic acid
into RNA is in the epidermis incubated i n
RPMI either alone or attached to its dermis.
Within this group in turn, the highest specific
activity of RNA is for epidermis incubated
alone at 37°C-the condition, interestingly
enough, which shows the greatest net loss of
RNA. The incorporation of ('<HIorotic acid into
the ASM is also greatest in epidermis cultured
alone in RPMI (Table 3).
For comparison, we have also studied the incorporation of (:iH) orotic acid into RNA and
ASM of normal mouse epidermis in vitro. We
have limited our study to normal epidermis
attached to its dermis, incubated a t 26"C,
either in STKM or RPMI, because these are the
conditions which give the best results for regenerating epidermis. In Table 4 it is seen that
the values for RNA, DNA, and ASM after the
3-hour incubation period at 2 6 T , in either sucrose TKM or RPMI, are quite close to the values seen in intact mice (Table 1).Table 4 also
indicates that the best incorporation of (:jH)orotic acid into RNA is in the RPMI medium.
Under both conditions of study, there is very
good preservation of histological detail (not
shown).
DISCUSSION
The best preservation of RNA is obtained
when the regenerating epidermis is incubated
attached to its dermis at 26°C in buffered
sucrose. Almost as good RNA preservation is
obtained in RPMI medium under similar conditions. Increasing the temperature to 37°C results in a considerable loss of RNA in the regenerating epidermis incubated i n either
STKM or RPMI. Incubating the regenerating
epidermis without its underlying dermis also
results in a significant decrease in RNA, which
becomes even more severe when the incubation
is done at 37°C. The DNA of the regenerating
epidermis is minimally affected by either increasing the temperature or incubating the
epidermis without its dermis. The amount of
acid soluble material as measured by the
number of A,,, units/g epidermis appears to rise
moderately in all conditions of incubation.
The incorporation of ('jH) orotic acid into
RNA in regenerating epidermis incubated in
vitro under conditions of maximal preservation
of RNA, that is when the regenerating epidermis attached to its dermis is incubated at
26"C, in either STKM or RPMI, is quite good.
However, the highest specific activity of RNA
in the regenerating epidermis occurs when the
epidermis is incubated alone at 37°C in RPMI
medium. Calculations indicate that this increased specific activity of RNA cannot be accounted for solely by the increase i n the specific
activity of the acid soluble pool. Interestingly,
incubation of epidermis alone in RPMI results
in the greatest loss of RNA. This raises the
possibility that the higher specific activity of
the RNA in the epidermis incubated in RPMI
medium at 37°C may be a n artefact, because
the RNA which was lost was the RNA which
had already been synthesized prior to labelling
the epidermis with ("H) orotic acid, and therefore, was not labelled. This would make comparisons of RNA specific activity with the specific activity of RNA under conditions in which
little, if any, RNA is lost difficult, because the
lower specific activity in the regenerating epidermis could be due to the fact that the cells of
the regenerating epidermis contain considerable amounts of unlabelled RNA, which would
lower the value for the specific activity of the
RNA. We, therefore, calculated the incorporation of H")(
orotic acid into RNA per mg DNA.
The cpm in epidermal RNA, when calculated
per mg DNA, show a considerable reduction in
RNAC
DNA
ASMd
RNA
DNA
ASM
Condition
cpm
mgRNA
CPm
A,=its
Cpm
mgRNA
CPm
A.,,,,=its
"Average r standard error of the mean.
"Number of determinations In parentheses
Epidermis and dermis
Epidermis alone
epidermis
of
2.50 ? 0.150 (8)
2.15 ? 0.130 (8)
56.2 t 5.60 (8)
2.75 t 0.150 (9)
2.15 -c 0.110 (9)
61.0 ? 4.40 (9)
0.25 M Sucrose - TKM
37°C
3.27 ? 0.470a (13P
2.98 ? 0.120 (13)
49.3 -c 3.60 (13)
4.38 t 0.140 (8)
2.75 i- 0.160 (11)
65.2 t 4.30 (11)
26°C
1.25 t 0.140 (5)
2.43 f 0.0070 (5)
61.7 -c 10.7 (5)
3.67 t 0.170 (10)
2.87 f 0.230 (10)
49.9 c 2.70 (10)
26°C
37°C
8,400 t1,300
(9)
11,700 i- 800
(6)
(8)
(8P
31,900t 5,000
22,900? 5,700
(8)
7,600 t 600
(9)
5,600 f 900
(9)
(5)
43,300 -c 12,000
(5)
32,400 ? 7,800
(10)
15,300 -c 1,600
(10)
57,700 t 12,200
9,800 f 800
(8)
26°C
37°C
0.25 M. Sucrose - TKM
18,500-c4,7OOd
26°C
RPMI
(6)
29,500 ? 9,100
53,400(6)
2 6,300
125,600 t 24,500
37°C
1.11? 0.080 (8)
2.45 ? 0.150 (8)
47.4 f 3.9 (8)
2.84 t 0.220 (8)
2.96 -c 0.230 (8)
65.8 ? 4.60 (8)
RPMI
TABLE 3 . Incorporation of (5:'H) orotic acid into RNA and ASM of regenerating female mouse epidermis 7 ahys after abrasion,
incubated in vitro for 3 hours
^Average 2 standard error of the mean.
"Numberof determinations in parentheses.
'RNA and DNA, mgig epidermis.
dASM, A,,,, uniWg epidermis.
Epidermis
and dermis
Epidermis
alone
Condition
of
epidermis
TABLE 2. The RNA, DNA, and ASM content of regenerating female mouse epidermis 7 days after abrasion incubated in vitro for 3 hours
4
E
0
2
U
4
5
5
z
0
3e
5
e
E
i
EPIDERMAL RNA IN VITRO
353
TABLE 4. Incorporation of (&'HI orotic acid into epidermal RNA and ASM, and the epidermal
RNA, DNA, and ASM content of normal female mouse epidermis plus dermis incubated
in uitrv far 3 hours
Measurement
0.25 M Sucrose - STKM
RPMI
RNA
(mgig epid.)
DNA
(mgig epid.)
ASM
(A,,,,units/g)
1.44 2 0.080
(8)
4.10 2 0.40
(8)
26.10 -t 1.70'
(4)
20,100 i 3,400
1.36 ? 0.110
(10)
4.10 ? 0.450
(10)
21.80 ? 0.440
(10)
220,000 2 9,100
(10)
61,700 ? 1,300
(6)
(10)
(8)"
32,600 2 2,600
"Average 2 standard error of the mean.
"Number of determinations in parentheses.
the regenerating epidermis incubated alone a t
ACKNOWLEDGMENTS
26°C or 37"C, but the incorporation of ("H)orotic acid into RNA is still higher in epidermis
This investigation was supported by NIH
incubated under conditions in which RNA is grant AM18219.
lost. Therefore, at least a portion of the inThe authors wish to thank Anthony Brigandi
creased specific activity of RNA is not simply for his expert help in this investigation.
due to the loss of unlabelled RNA.
The effects of 3-hour incubation on normal
epidermis was studied only for normal epiLITERATURE CITED
dermis attached to its dermis a t 26"C, in either Argyris, B.F. (1977) Suppressor cells in the spleen of tumorSTKM or RPMI, because these were the condiallosensitized mice. Cancer Res., 37t3390-3399.
tions which gave the best preservation of R.NA Argyris, T.S. (1978) Epidermal growth and ribosomal RNA
accumulation in regenerating mouse epidermis following
in regenerating epidermis. Normal epidermis
abrasion. J. Invest. Dermatol., 70:267-271.
attached to its dermis, incubated a t 26°C in Argyris,
T.S. (1977) Kinetics of regression of epidermal
either STKM or RPMI, shows no loss of RNA
hyperplasia in the skin of mice following abrasion. Am. J.
Pathol., 88,575582.
and DNA, and no significant change in the
amount of acid soluble material. There is also Argyris, T.S. (1976) Unbalanced RNA accumulation in regenerating mouse epidermis following abrasion. J. Invest.
good preservation of histological detail. IncorDermatol., 67: 7 18-722.
poration of ("1 orotic acid into RNA is higher DeLapp, N.W., and M.A. Karasek (1976)Importance of pyin the RPMI medium than in buffered sucrose,
rimidine nucleotide salvage pathways for DNA synthesis
in skin. J. Invest. Dermatol., 66:30&312.
but the latter is quite adequate.
L.M., T.S. Argyris, and G.B. Gordon (1977) EpiIn conclusion, this study demonstrates that DeYoung,
dermal ribosome accumulation during two-stage skin
when studying RNA metabolism of mouse epitumorigenesis. Cancer Res., 37:38&393.
dermis in vitro, it is important to determine the Flamm, W.G., M.R. Barnejee, and W.B. Counts (1966) Topical application of actinomycin D on mouse skin: Effect of
effects of the incubation period on the RNA
the synthesis of ribonucleic acid and protein. Cancer Res.,
content. Our study demonstrates that the RNA
26: 134S1360.
content can significantly decrease, even within Mueller, S.N., and T.S. Argyris (1977) Patterns of growth
and ribosome accumulation during 3-methylcholana 3-hour period, under certain in vitro condithrene-induced epidermal hyperplasia. Cancer Res.,
tions, which show little change in DNA con37t3400-3405.
tent, good incorporation of (3H)orotic acid into Mueller,
S.N., and T.S. Argyris (1975) Free and membraneRNA, and very good preservation of epidermal
bound ribosomes in normal and methylcholanthrenetreated
mouse epidermis. Lab Invest., 32.209-216.
histological details.
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